Unbranched Poly(β-L-malic acid) (PMLA) in FIG. 1 is a biopolymer produced from renewable feedstocks (1). The polymer consist of units L-malic acid esterified via the carboxylic group in beta-position and the hydroxyl group. PMLA can also be synthesized chemically (1); however, chemically synthesized PMLA shows lower purity and partial racemisation. The molecular weight (Mn, number-averaged molecular weight) of the biopolymer ranges from 3,000 to 300,000 with the highest proportion at 50,000-100,000 (2). Plasmodia of the Physarum family produce PMLA in excess of their physiological needs and secrete large amounts into the culture medium (3). Published producers for the strains M3CII and M3CVIII (ATCC 204388 and ATCC 96951) of Physarum polycephalum have been reported by us, but any other Physarum species can be active in PMLA production as claimed here [see references in (1)]. The yield by conventional production methods in culture flasks is less than 1 g/liter of culture broth.
Some filamentous fungi produce short-chain PMLA (branched and of approximately 10,000 molecular weight) that is conjugated to poly(glucan) [(4) and references in (1)] involving activities of secreted enzymes to remove PMLA from the poly-glucan residues (1). This kind of PMLA preparation cannot be purified to chemical homogeneity because of uncontrolled amounts of covalently attached sugar moieties.
PMLA from plasmodia is unbranched, devoid of any covalently attached other molecule, and easily purified to chemical homogeneity. Physarum produces agglutinins of low molecular mass. These secreted proteins purify by a characteristic pathway that is different from the one used for the purification of polymalic acid. PMLA preparations contain endotoxin originating from yeast extract included in the fermentation broth. The contaminant has not been removed.
Polymalic acid is produced by the species of the Physarum family, in particularly by the yellow strains of Physarum polycephalum, and secreted into the culture broth. The broth will contain then small amounts of peptides/proteins, polysaccharides (slime), pigments, and various inorganic salts (1) that must be removed by purification.
The polymer consists of units malic acid esterified between their hydroxyl group and the carboxylic group in β-position (FIG. 1). The carboxyl groups in α-position are free to ionize with a pKa of about 4 to yield polyanion at neutral pH. The acid form and the anionic form of PMLA differ by solubility in organic solvents. Both forms are highly soluble in water, but only the acidic form dissolves in acetone and several other organic solvents.
The polyanion is precipitated with ethanol, especially in its form of the calcium salt. PMLA binds with high affinity to polycationic (chromatographical) material from which it is eluted with queopus solutions containing >0.5 M NaCl or any other cation.
The binding of the polyanion to polycationic material, the solubility of PMLA in the acid form in acetone, and the ready precipitation of the calcium salt with ethanol allow the efficient isolation of highly purified polymalic acid after conversion of the calcium-salt into PMLA-acid.
PMLA in the culture broth has a molecular weight 30,000 to 300,000 and the inhomogeneity corresponds to a polydispersity value (Mw/Mn) of 2 (1). PMLA of a narrow distribution in molecular weight can be produced by gel permeation chromatography.
Because of the instability of the ester bond, exposure to solutions of pH<5 and pH>9 must be avoided to keep spontaneous hydrolytic cleavage at a minimum (7). PMLA is also enzymatically hydrolysed in the culture broth at a maximal rate when pH is 3-4 (9).
Polymalate Producing Organisms (FIG. 2)
Polymalate is produced by plasmodia of the Physarum family. Plasmodia of the Physarum family are multinuclear, large, at a late growth stage slimy, unicellular organisms, commonly called slime molds (5). Plasmodia of Physarum polycephalum and of other family members convert D-glucose to PMLA (6). Several Physarum strains have been shown to secrete PMLA into the culture broth. Among these are strains M3CVII and M3CVIII. To date all yellow colored strains tested were found to be good PMLA producers (FIG. 2), and it is evident that all yellow strains are good producers of this polymer. It is proposed to generate new such strains of the Physarum family and of other myxomyceteae strains by cross breeding and selection for PMLA synthesis following an automated chemical testing of the culture broth (1). Viable plasmodia can be stored in the form of resting cells, so-called spherules, which are the dormant and highly durable during may years (1). Spherules can be prepared and activated to plasmodia by following existing methods.
Prior Art PMLA Culture Methods
For simple PMLA production, shaken cultures in 2 liter indented Erlenmeyer flasks of 300 mL production medium are inoculated with 5 ml cells of a starting culture. After four days at 24° C. in the dark, the culture broth is removed and is processed for the isolation of the polymer.
The PMLA production medium contains the following ingredients (g/liter):                10 D-glucose        10 Bactotrypton (DIFCO, Germany)        1.5 yeast extract (DIFCO)        3.5 citric acid monohydrate        2.0 KH2PO4         0.6 MgSO4-heptahydrate        0.085 MnCl2-tetrahydrate        0.085 FeSO4-hepthydrate        0.035 ZnSO4-heptahydrate        0.0025 hemin        Deionized water to make.        
Solutions in the absence of D-glucose and hemin are brought to pH 4.3 with 5 M NaOH and are then sterilized (autoclaved) at 120° C. The remaining ingredients at 10 times concentration are sterilized separately at 120° C. and then added to the other ones. Maintenance culture are routinely grown for 2 days at 24° C. in 100 ml medium (500 ml indented flasks).
Precultures (for inoculation) and production cultures are grown in the same medium at the same conditions.
Spherules
Spherules is a durable resting stage cell form of the plasmodium and can be kept for many years on filter strips without loosing viability. A piece of filter paper containing sperules (e.g. strain M3CVII) can be placed onto a 1.5% agar (10 cm petri dish) containing 2-fold diluted culture medium (for microplasmodia, see below) and left at 24° C. in the dark. After 2-3 days, plasmodia start to hatch. A growing plasmodium (a flat cell) is then transferred to fresh agar and grown until it almost completely covers the agar surface. The cell is detached from the agar and transferred into 100 ml culture medium (500 ml dented Erlenmeyer flask) and cultured at 24° C. in the dark on a Gyro-Shaker (G10 New Brunswick) at 100-180 rounds per minute (rpm) (plasmodia require lots of oxygen). For optimal culturing, every 2-3 days, a portion of 5-10 ml settled cells is transferred to fresh culture medium.
Spherules are prepared to have a reliable stock supply of plasmodia for future use. Spherules can be stored at a dry and cool place for many years. For their preparation, a 2-day-culture of microplasmodia is allowed to settle to the bottom of the flask. The supernatant is decanted quantitatively. One hundred milliliterts of spherulization medium is added and shaking continued at 24° C. After three to four days orange colored spherules are formed. They are centrifuged, resuspended in a small amount of spherulization medium and placed in small aliquots on sterile filter paper. After they have dried, they are stored at 4° C. in the refrigerator.
Spherulization Medium (g/liter):
4.0citric acid0.09FeSO4 × 7H2O0.6MgSO4 × 7H2O1.2CaCl2 × 2H2O0.085MnCl2 × 4H2O0.035ZnSO4 × 7H2O4.4KH2PO4
The aqueous solution is adjusted with 30% KOH to pH 3.8 and sterilized for 20 min at 120° C.
Analytic methods for asseying PMLA (reference 2):
Hydroxamate/Fe(III)-color assay (For rapid, but less sensitive measurements):    Adjust the sample to 320 TI with distilled water and mix with 160 μl of reagent A. Add 160 μl of reagent B. Allow the reaction to proceed for 10-15 min. Mix with 160 μl of reagent C, and read the absorbance at 540 nm wavelength (1 mg/ml PMLA=2.5 A540).
Reagent A=10% (w/v) hydroxylammonium chloride.
Reagent B=10% (w/v) NaOH.
Reagent C=5% (w/v) Fe(III)Cl3 in 12% (v/v) HCl.
Malate dehydrogenase assay (For sensitive measurements):
Hydrolyze the sample PMLA for 2 h at 100° C. in the presence of 2 M H2SO4. After careful neutralization with 5 M NaOH, L-malate (besides a few percent fumaric acid) is measured photometrically at 340 nm wavelength in equivalents of NADH formed in the presence of 60 units/liter malate dehydrogenase in the presence of 40 mM NAD+, 0.76 M glycine and 0.5M hydrazine hydrate (0.5 M), pH 9.0 for 30 min at 37° C. The absorbance of NADH is standardized by repeating the enzymatic reaction with known amounts of L-malate.
Enzymatic Test for Verification of the Linear Structure of PMLA
The unbranched nature of polymalic acid can be verified by its enzymatic exhaustive cleavage to L-malic acid by Polymalatase (14). PMLA with branching points or chain substitutions is not cleaved beyond cbranching points. Branching is experimentally detected by comparing the experimental content of malic acid, using any one of the quantitative malic acid detection assays, with the theoretical content of malic acid calculated on the basis of the sample weight.
Prior Art Poly(Malate) Isolation Technique
For purification, culture broth is passed through DEAE-Cellulose (8) followed by washing over a Büchner funnel with 15 volumes of 10 mM sodium phosphate buffer pH 7, containing 0.3 M NaCl that removes proteins and most of the polysaccharides and pigments. PMLA is eluted with 6 volumes of 10 mM sodium phosphate solution of pH 7 containing 0.7 M NaCl. After proper dilution, the polymer is adsorbed on fresh DEAE-cellulose material and subjected to chromatography with 7 volumes buffer containing a gradient of 0.2-1.5 M NaCl. This step is repeated, then the PMLA-containing fractions precipitated with 70% (v/v) ethanol in the presence of 0.2 M NaCl and concentrated by freeze-drying. Salt is completely removed by molecular sieving over a Sephadex G25 fine. PMLA, sodium salt, is converted into the acid form over Amberlite 120 H+ then freeze-dried and dissolved in anhydrous acetone. Insoluble material is removed and acetone evaporated under reduced pressure to yield solid, highly purified, colorless PMLA-acid. The prepared material is not hygroscopic and shows crustallization. The yield is 20-30% of the amount of PMLA in the culture broth. The labor time is approximately 2 weeks.
Technical Problems Of The Prior Art PMLA Production And Isolation Methods
The prior production method suffers from low production capacity and bad reproducibility affording a high expenditure of labor, space, glass ware, and other materials, and is not applicable to biotechnology. Growth in 500 mL culture broth in indented Erlenmeyer flasks is not commercially realizable. Results of culturing on shaker tables are not reproducible because of uncontrolled pH, aeration, agitation and prolonged incubation. Multiple handling in the absence of sterilization results in frequent and high levels of contamination, or loss of culture and low PMLA production
The production method has limited capacity not eceeding 10 liters of culture broth, allowing a maximum output of 2-3 g PMLA.
Purification leads to excess inorganic salt (NaCl, KCl, or CaCl2) in polymalate preparations that must be totally removed by repeated gel filtration before acidification. Failure will provoke low quality of polymalic acid by containing HCl that will cause hygroscopicity and rapid degradation of the polymer. Extended exposure to DEAE-cellulose and Amberlite 120 H+ leads also to cleavage and loss of high molecular weight PMLA. Preparations contained polypeptides such as endotoxins.
Objects of the invention
                It is an object of the present invention to define reproducible conditions for a culture broth optimal for high molecular weight PMLA production;        It is a further object of the present invention to present al large scale production of PMLA allowing scale up PMLA production in closed bioreactors of virtually any size;        It is another object of the present invention to allow plasmodia cultivation under control and repeated adjustment of temperature, pH, aeration, agitation as well as suppression of foam and avoidance of contamination; and        It is a still further object to establish perfect culture conditions that avoid cell death and physical cell lysis by providing unharsh conditions for growth and harvesting while reducing spontaneous and enzymatic cleavage of produced PMLA.        It is an additional object to provide a simplified isolation method on a large scale that can be readily scaled up;        A still further object of the invention is to increase capacity and throughput of the polymalic acid isolation method at a minimum of time;        Another object is to provide improved removal of salt from the polymalate preparation before conversion into polymalic acid over Amberlite 120 H+in order to avoid contaminatiin by HCl derived from salt; and        An additional object of the present invention is to convert PMLA by removal of solvent water within a minimum of time to avoid HCl-catalysed degradation after acidification.        Another object of the present invention is to purify polymalic acid free of agglutinins and endotoxins.        